Vascular thrombosis is a cardiovascular disease indicated by the partial or total occlusion of a blood vessel by a clot containing blood cells and fibrin. In arteries, it results predominantly from platelet activation and leads to heart attack, angina or stroke, whereas venous thrombosis results in inflammation and pulmonary emboli. The coagulation of blood is the result of a cascade of events employing various enzymes collectively known as activated blood-coagulation factors. Heparin, a powerful anticoagulant has been used since the late 1930's in the treatment of thrombosis. In its original implementation, tolerance problems were noted and so reduced dosage was suggested to reduce bleeding and improve efficacy. In the early 1970's, clinical trials did indeed indicate acceptable tolerance was obtainable whilst still preserving antithrombotic activity. Unfractioned heparin (UFH) is primarily used as an anticoagulant for both therapeutic and surgical indications, and is usually derived from either bovine lung or porcine mucosa. Among the modern uses of unfractioned heparin are the management of unstable angina, an adjunct to chemotherapy and anti-inflammatory treatment, and as a modulation agent for growth factors and treatment of hemodynamic disorders.
In the late 1980's, the development of low molecular weight heparins (LMWHs) led to improvements in antithrombotic therapy. LMWHs are derived from UFH by such processes as; chemical degradation, enzymatic depolymerisation and γ-radiation cleavage. This class of heparins has recently been used for treatment of trauma related thrombosis. Of particular interest is the fact that their relative effects on platelets are minimal compared to heparin, providing an immediate advantage when treating platelet compromised patients. The degree of depolymerisation of UFH can be controlled to obtain LMWH of different lengths. Dosage requirements for the treatment of deep vein thrombosis (DVT) are significantly reduced when employing LMWH as opposed to UFH, although in general the efficacy of both therapeutics seems to be comparable. In addition, LMWH can be effective as an alternative therapeutic for patients who have developed sensitivity to UFH. Unfortunately, there has recently been a great deal of concern in the use of LMWH due to the perceived potential for cross-species viral contamination as a result of the animal source of the parent UFH.
One way of avoiding the possibility of cross-species contamination, is to prepare heparins by chemical synthesis. This method would also provide the opportunity to develop second generation heparins or heparinoids that can be tailored to target particular biological events in the blood coagulation cascade.
An investigation to determine the critical structural motif required for an important binding event in a coagulation cascade involving heparin, dates back to the 1970's. Some structural features of heparin were defined, but the binding domains of interest remained essentially undefined. Research conducted by Lindahl and co-workers (Proc. Natl. Acad. Sci. USA, 1980, Vol. 77, No. 11, 6551-6555; J. Biol. Chem., 1981, Vol. 256, No. 5, 2389-2394) and separately by Choay and co-workers (J., Annals New York Academy of Sciences, 1981, 370, 644-649) eventually led to the determination that a pentasaccharide sequence constituted the critical binding domain for the pro-anticoagulant cofactor, antithrombin III (AT-III). After determination of the critical heparin sugar sequence, complete chemical syntheses were embarked upon to further prove the theories. Complete syntheses of the pentasaccharide binding domain were completed at similar times by Sinay and co-workers (Pierre Sinaÿ, Jean-Claude Jacquinet, Carbohydrate Research, 132, (1984), C5-C9) and by Van Boeckel and co-workers (C. A. A. van Boeckel, T. Beetz, J. N. Vos, A. J. M. de Jong, S. F. van Aelst, R. H. van den Bosch, J. M. R. Mertens and F. A. van der Vlugt., J. Carbohydrate Chemistry, 4(3), 1985, 293-321).
Significant difficulties were encountered during both these reported syntheses. The synthesis by Van Boeckel and co-workers provided a method on reasonable scale (156 mg's of final product) and with improved yields compared to the Sinay synthesis, but still only provided an overall yield of 0.22%, (compared with 0.053% for the Sinay synthesis). One particular problem encountered during the final deprotection, was the intermolecular reaction of the hemiacetal (the reducing end functionality of the sugar), which led to the formation dimers and trimers. To reduce the likelihood of this occurring, an α-methyl glycoside of the pentasaccharide was synthesized. The structures of interest are represented in Structure 1, wherein I represents the hemiacetal form, and II represents the α-methylglycoside form.

As mentioned, studies have determined that the significant biological event in preventing thrombosis is the binding of a pentasaccharide sequence (Ann. NY Acad. Sci., 1981, 370, 644-649) of heparin, to heparin cofactor antithrombin III (AT-III). As well as pentasaccharide I, the important derivative III has also been prepared by total synthesis (Biochem. Biophys. Res. Commun., 1983, 116, 492-499). The following patents display some relevance to the present invention. U.S. Pat. No. 4,401,662 claims composition of matter on the pentasaccharide AT-III binding sequence of heparin as does U.S. Pat. No. 4,496,550. EP 0,084,999, U.S. Pat. No. 4,818,816, U.S. Pat. No. 7,541,445 and U.S. Pat. No. 8,114,970 detail synthetic methodologies towards pentasaccharide I, and derivative II.
Current synthesis processes of heparinoids, especially for preparation of disaccharides and trisaccharides, still have problems in the following steps with reference to FIG. 1.
Imidate formation: disaccharide-2→disaccharide-3 or trisaccharide-2→trisaccharide-3
Well-known imidate formation reaction was carried out with organic base, 1,8-Diazabicyclo[5,4,0]undec-7-ene (DBU), and trichloroacetonitrile (TCA) as limiting reagent in an organic solvent to obtain imidate. The resultant compound was separated and purified with silica gel column. This reaction requires large amount of organic solvent and time to concentrate to obtain the product.
Glycosylation: Glycosylation of Disaccharide-2 and Monosaccharide C to produce Trisaccharide-1
In Disaccharide-2 molecule, the substituent at the second C atom of Monosaccharide D is OBn. It is prone to form α epimer in general glycosylation reaction without neighboring group effect.
Desilylation: Trisaccharide-1→Trisaccharide-2
In general desilylation reaction, especially for bulky protecting group, tert-butyldimethylsilyl group (TBDMS or TBS group), tetrabutylammonium fluoride (TBAF) was used to remove TBS protecting group, and then separated and purified with silica gel column. This reaction also requires large amount of organic solvent and time to concentrate to obtain the product. It is not easy to remove TBAF effectively in the resultant compounds so that the purified product is difficult to store because of degradation and deterioration.
There is still a need to develop simple, convenient, and efficient synthesis process for preparation of disaccharides and trisaccharides of heparinoids.